Investigating the role of the T1 domain in the gating mechanism of KV1.1 channels

KV1.1 channels are encoded by the KCNA1 gene and often found as hetero-tetramers with other KV1 α-subunits. They are widely expressed in the nervous systems and responsible for a variety of physiological functions, ranging from regulating action potentials to involvement in cell apoptosis. Despite decades of research, there is still much that is unknown about the physiological role of some of KV1 α-subunit domains in their gating mechanisms. An example of this is the tetramerisation domain (T1) domain, located at the cytoplasmic N-terminus of the KV ion channels’ α-subunits.

This project examines how T1 domain tetrameric stability affects gating properties of the KV1.1 channels. For this, amino acid residues that were predicted to affect the T1 domain stability were computationally identified. KV1.1α channel constructs with these mutations were made and functionally characterised using two electrode voltage clamp (TEVC). Out of these, mutations at residue L46 were shown to have significant effects on the voltage dependent gating properties of KV1.1 channels. These were L46F and L46D, which were predicted by in-silico analysis to cause de-stabilisation and increased stabilisation of the T1 domain respectively.

Biochemical mixing assays, as well as thermal melt experiments were then used to assess the effect of L46 point mutations on the T1 domain protein stability. They all confirmed the predictions of the in-silico analysis stated above. Furthermore, NMR spectroscopy of these mutants found chemical shift changes occur at T1 domain subunit interfaces following L46 point mutations which taken together with functional and biochemical data presented here, strongly supports the impact of L46 point mutations on T1 domain tetrameric stability.

Together, these data show changes in the T1 domain stability impact KV1.1 channel gating properties, which is consistent with the T1 domain and its stability playing a physiological role in the gating mechanism of KV1.1 channels.